ABSTRACT Alzheimer?s disease (AD) is a debilitating neurological disease and the leading cause of dementia. The pathophysiology of AD includes neuronal loss and is characterized by the buildup of amyloid plaques and neurofibrillary tangles, leading to extensive study of these disease processes. Recently, evidence has suggested that blood-brain barrier (BBB) dysfunction may contribute to the progression and severity of AD. However, the mechanisms of BBB dysfunction in AD remain to be elucidated. The BBB acts as a signaling and transport interface between the blood and brain, and with its very low permeability and a wealth of molecular transport systems, the BBB helps regulate the extracellular fluid of the brain. While brain endothelial cells mediate these BBB functions, the BBB is greatly influenced by endothelial cell interactions with supporting cells of the neurovascular unit (NVU), including astrocytes, pericytes and neurons. Currently, it is unclear which human BBB functions are affected in AD, what causes these BBB changes, and which NVU cell types are responsible. BBB dysfunction in AD could be caused by genetic factors such as ApoE allele, a major AD risk factor, or may be secondary to neurodegenerative disease processes. In this proposal, we will investigate both possibilities. First, by deploying a powerful and innovative approach for modeling human disease using induced pluripotent stem cell (iPSC) technology, we will investigate the impact of ApoE allele on BBB function. We have recently demonstrated that it is possible to derive each of the key NVU cell types from patient- sourced iPSCs, and that these models can be used to better understand BBB dysfunction in genetic human disease. Here, the iPSC-derived NVU model will be used to investigate ApoE allele combinations to determine their effects on BBB barrier, transport and immune functions and to identify the key NVU cell type driving the observed effects. Next, to investigate the possibility that BBB dysfunction is secondary to disease processes, we will identify the molecular changes in human AD brain endothelial cells and then examine their function in the iPSC-derived NVU model. Brain endothelial cells will be isolated from brain tissue of AD patients and state- of-the-art multiplex proteomic methods used to identify proteins that are differentially abundant in AD brain endothelium. We will then use gene editing techniques to create iPSC lines in which we can modulate expression of the differentially regulated genes and evaluate their effects on BBB function using the multicellular iPSC-derived NVU model. A better understanding of the sources and forms of BBB dysfunction in AD will yield new mechanistic insights into AD disease progression, and suggest new avenues for therapeutic intervention.